DESCRIPTION: Our discovery of a novel enzyme in mycobacteria (coenzyme-F420-dependent glucose-6-P dehydrogenase), and our recent demonstration that M. smegmatis mutants lacking this enzyme grow poorly and have increased sensitivity to oxidative stress suggest that F420 is important for Mycobacterium fitness. F420 involvement in Mycobacterium metabolism was unexpected, since it is an electron-transfer coenzyme seldom found in Bacteria, but commonly found in Archaea. Mycobacteria cause several serious diseases, including tuberculosis, leprosy, and AIDS opportunistic infections. If F420 is important for virulence, we think that understanding F420 metabolism and biosynthesis will provide clues on how to develop new classes of drugs active against mycobacterial disease. We propose to determine the genetic basis for coenzyme F420 biosynthesis in Mycobacterium, and to define the importance of this coenzyme for growth, oxidative stress response, and virulence. This will be achieved by isolating M. smegmatis mutants that cannot make F420, cloning and sequencing the genes affected, using this information to create F420-minus Mycobacterium tuberculosis mutants, evaluating the vitro properties of all mutants, and deducing as much as possible concerning the role of each gene. The virulence of M. tuberculosis mutants will be examined in macrophage and animal models. The main hypotheses are that: F420 is important to mycobacteria as a redox coenzyme in growth, protective oxidative stress response, or other aspects of metabolism; F420 is important for M. tuberculosis virulence; the F420 biosynthesis genes will be largely consistent with the proposed biosynthesis pathway in methanogenic Archaea.